以磷酸酰胺Phaseolotoxin为模型的微生物天然产物氮磷键生物合成研究

Phosphoramidate natural products refer to a group of Nitrogen-Phosphorus bond (N-P bond) containing compounds produced by living organisms. Attribute to the structure similarity with phosphate biomolecules, it was liable to be misrecognized and bound by certain enzymes in vivo, while could not be catalyzed successfully. Phosphoramidate natural products show diverse bioactivity by inhibiting some essential enzymes of the target organisms, such as Phaseolotoxin is a broad-spectrum biopesticide. Thus far, only fourteen phosphoramidate natural product molecules have been discovered and our understanding about its N-P bond biosynthesis is really limited. Phaseolotoxin, which contains the most N-P bonds number, was selected as a model for N-P bond biosynthesis study in this project. Gene interruption, biosynthetic pathway rebuilding, and biosynthetic intermediates characterization would be employed to identify genes directly related to N-P bond formation. Then, in vitro biochemical experiments would be conducted to address N-P bond biosynthetic pathway. Key reaction mechanisms would be deciphered by enzymology and structural biology experiments. Based on the general understanding of N-P bond formation mechanism, genome mining and combinatorial biosynthesis of phosphoramidate natural products would be carried out as well.

磷酸酰胺天然产物是生物体产生的含有氮磷键的化合物，因为其结构与磷酸化的生物小分子相似，所以容易被细胞内某些酶错误的识别、结合，但是不能被有效的催化。磷酸酰胺天然产物通过抑制靶标生物生命活动某些必须酶的功能，表现出各种各样的生物活性，比如菜豆菌毒素（Phaseolotoxin，PHT）就是一种广谱的生物除草剂。迄今为止发现的磷酸酰胺天然产物仅14个，对于磷酸酰胺天然产物氮磷键的生物合成机制认识也非常有限。PHT含有最多的氮磷键数目(三根)，本项目计划以PHT为模型，研究氮磷键的生物合成机制。我们计划通过基因中断和合成途径重构，鉴定中间体，确定PHT氮磷键合成直接相关的基因。然后通过体外生化实验解析PHT氮磷键的生物合成过程，辅以酶学和结构生物学手段揭示关键反应的催化机制。在对氮磷键的生物合成形成规律性认识的基础上，我们将开展基因组挖掘和组合生物合成，以便发现和创造更多的磷酸酰胺天然产物。

菜豆菌毒素Phaseolotoxin是植物病原菌丁香假单胞菌Pseudomonas syringae pv. phaseolicola产生的磷酸三酰氨天然产物，含有特殊的N-P-N-S杂原子结构单元。Phaseolotoxin抗性基因argK编码的蛋白质ArgK是一个对Phaseolotoxin不敏感的氨甲酰基转移酶，可以替代Phaseolotoxin的作用靶标蛋白质ArgF的功能。本研究证明ArgK具有底物宽泛性，既可以以鸟氨酸为底物生成瓜氨酸，发挥ArgF的活性；又可以以“鸟氨酸-丙氨酸-高精氨酸”三肽为底物，争夺Phaseolotoxin的生物合成中间体，调控Phaseolotoxin的产量。丁香假单胞菌只在18℃产生Phaseolotoxin，而在其最适生长温度28℃不产生Phaseolotoxin。本研究表明这种温度依赖性与抗性基因argK无关，而是与其生物合成基因簇中的脂肪酸去饱和酶编码基因desI直接相关。Phaseolotoxin生物合成基因簇共23个基因，本项目对其中的16个进行了功能鉴定，证实PhtA和PhtL与氮磷键的形成直接相关。该基因簇在恶臭假单胞菌中异源表达可以合成Phaseolotoxin，Nδ(N’-sulfo-diaminophosphinyl)-ornithine是Phaseolotoxin的生物合成中间体。Phaseolotoxin的磺酸基团来源于3′-磷酸腺苷-5′-磷酰硫酸，磷原子来源于ATP，氮磷键的形成依赖于磺酸化修饰。磷酸二酰氨中间体首先在PhtL蛋白上形成，然后转到鸟氨酸的德尔塔氨基上，形成磷酸三酰氨，PhtL蛋白质His600氨基酸残基在氮磷键行成中起到决定性作用。通过基因组挖掘，我们还找到了新的磷酸酰胺天然产物生物合成基因簇；对氮硫键、碳硫键等杂原子成键反应进行拓展研究，揭示了新型天然产物碳硫键形成机制。